Psychological stress-induced accelerated colonic transit in rats involves hypothalamic corticotropin-releasing factor

1993;104:716-723

GASTROENTEROLOGY

Psychological Stress-Induced Accelerated Colonic Transit in Rats Involves Hypothalamic Corticotropin-Releasing Factor HUBERT

MijNNiKES,

BEATE

G. SCHMIDT,

and YVETTE

TACHi

Center for Ulcer Research and Education, Veteran’s Affairs Wadsworth Medical Center; Department of Medicine and Brain Research Institute, University of California at Los Angeles, Los Angeles, California

Background: Brain corticotropin-releasing factor (CRF) is involved in stress-induced accelerated coionic transit. Brain sites of action of CRF to stimulate colonic transit were investigated in conscious fed rats. Methods: Bilateral guide cannuiae were chronically implanted into the paraventricular nucleus of the hypothalamus (PVN) or central amygdala for peptide microinjection and a catheter into the proximal colon to measure colonic transit. Results: CRF (0.6 nmoi/rat) injected into the PVN reduced coionic transit time by 84% and stimulated fecal pellet output 20-fold, whereas CRF injected into sites outside of the PVN or the central amygdaia had no effect. CRF stimuiatory action was prevented by chiorisondamine, and atropine methyl nitrate but not by bretyiium. The stress of avoiding water by standing on a small cube reduced colonic transit time by 75% and increased fecal output by 7-fold. Bilateral microinjection of CRF antagonist, a-helical-CRF,_,, , into the PVN abolished the colonic response to stress. The CRF antagonist had no effect on basal coionic transit time in nonstressed rats. Conclusions: Psychological stress-induced stimulation of coionic motor function in fed rats involves CRF pathways in the PVN.

G

rowing

evidence

indicates

cotropin-releasing

that endogenous

factor (CRF)

corti-

in the brain plays

stress-induced alterations of gastromotor function. ‘s2CRF injected into the cerefluid (CSF) mimicked the effects of various

like immunoreactive binding

of CRF into the PVN

tric

acid

colonic

secretion transit

prevented

restraint

emptying rats.13

and

markedly the role

of CRF

avoidance

study,

nucleus

of the vagus

of gastric

been

in fasted

reported

and motor

we further brain

connections

to

activity

in

investigated

sites in mediating of colonic

fed rats. In addition

the central

multiple

bilateral the PVN

transit

stimulation

PVN, we investigated

amygdala,

with

which

the dorsal

and contains

moto the has

motor

a rich peptidergic

innervation.16

Materials and Methods Male Sprague-Dawley IN) weighing controlled

300-350

(12-hour (22 f 2’C).

to housing

cages. Postsurgery,

rats (Harlan,

g were housed

illumination

acclimated

function. 5-8 Brain sites whereby exogenous and endogenous CRF acts to elicit such changes have recently been located in the paraventricular nucleus of the hypothalamus (PVN). Subpopulations of neurons in the PVN are unique among other hypothalamic neurons in their ability to project directly to medullary and spinal preganglionic neurons controlling the activity of the autonomic nervous system.g Abundant CRF-

transit

in conscious

of

models of psychological to an aversive stimulus

at specific

unique

delay

fear have

stress-induced

tor function

into

of colonic

colonic

rats.5”5 In the present

of gas-

stimulation

antagonist

stress-induced

conditioned

increase

and

rats.‘3*‘4 Moreover,

Recently, two experimental stress including avoidance (water)

Microinjec-

inhibition

emptying

and stimulation

ity, and temperature

into the CSF prevented of gastrointestinal motor

induced

of the CRF

intestinal brospinal

lical CRF3_41, injected stress-induced alterations

and

in fasted

microinjection

and fibers as well as CRF

in the PVN.“‘-‘2

tion

a role in mediating

stressors in inhibiting gastric emptying and stimulating colonic motor function through autonomic patha-heways in rats. 3-5 In addition, the CRF antagonist,

neurons

sites are found

light/dark Before

conditions

animals

under

for

Purina

Laboratory

the beginning

of the experiments.

of

cycle), humidsurgery,

1 week

were maintained

with ad libitum

Indianapolis, conditions

rats were in colony

in single cages

chow and tap water up to The

experiments

were

started

between

8:30 AM and 9:30 AM, and rats were

prived

of solid food at that time but had access to water.

de-

Surgery In rats anesthetized mg/kg,

intraperitoneally

with a mixture (IP);

Fort

of ketamine

Dodge

(75

Laboratories,

Abbrevlatlons used in this paper: BSA, bovine serum albumin: CRF, corticotropin-releasing factor: CSF, cerebrospinal fluid; IP, intraperltoneally; PVN, paraventrlcular nucleus of the hypothalamus. 0 1993 by the American Gastroenterologlcal Association 0016-5065/93/$3.00

March

COLONIC

1993

Fort Dodge, tion,

IA) and xylazine

Shawnee,

(5 mg/kg

KS), a silicone

1.7 mm) was chronically

implanted

1 cm distal from the cecocolonic fixed at the colonic subcutaneously cured

at the animal’s

central

and Watson.”

The cannulae

surgery,

animals

and

were

anchored

affixed

by dental

to the skull.

housed

ce-

After

nmol/rat)

of microinjection over

cannula below mine

(Plastic

VA) Then,

red was injected chronic

into the proximal catheter.

to monitor

the experiment mol/L

Torrance,

in 0.1% bovine

saline.

CA) was dissolved microinjection.

methyl

chlorisondamine,

carmine

red (Sigma

Alcian

in 0.15 mol/L

water

St Louis,

Inc.,

warmed

blue 8GX,

bretylium

Chemicals,

(BSA)

(Bachem,

into sterile

37°C just before nitrate,

serum albumin

a-helical-CRF,,,

brain site induce

immediately

tosylate, MO)

at

atropine and

were

dis-

or two different interval

colonic

Preliminary

Measurements Carmine

red (1.2 g/100

dye, was injected Colonic

between

the catheter

transit

carmine

followed

was evaluated

tored

by counting

When

diarrhea

pellet. pellets

occurred,

as the time

Fecal pellet every

by a 0.2-mL

in the proximal interval

and the discharge output

30 minutes

each diarrhoeal

equivalent

a nonabsorbable

positioned

red administration

the first red colored

was considered

mL saline),

in 0.2 mL volume

saline flush through colon.

of Colonic Transit

of

was moni-

for 24 hours.

bowel

movement

When mg/kg,

experiments

into the brain

as vehicle

or peptides.

cardial perfusion served

the visualization

data analysis.

was removed

after performing site. When

The criteria

based on the radius

of and

track was considin the

were only unilaterwere excluded

for including

microinjections

and con-

Brain sections

within

of spreading

from

values in data analy300 /trn from under

transit

time.

and CRF at one

in a randomized

fashion.

The

on the same animal

of rats from

the first experi-

to bilateral

into the PVN. minutes,

rats

microinjec-

When

were

colonic

subsequently

placement

of cannulae

assessed by histological

in the PVN

(which

examination

was fur-

at the end of the

experiment). Atropine

methyl

nitrate

(0.1 mg/kg),

mg/kg),

and bretylium

(15.8 mg/kg)

minutes

before

microinjection

bilateral

chlorisondamine,

injected

(3 IP 15

of CRF (0.6 nmol/

(0.1% BSA) into the PVN.

of atropine,

ditions3y’*

chlorisondamine were

and

The schedule bretylium

and were

studies showing muscarinic, ganglionic, blockade, respectively, under these con-

After peptide

or vehicle

microinjection,

the car-

mine red dye was injected into the proximal colon and coIonic transit time and fecal pellet output were monitored for 24 hours.

after trans-

the dye was detected animals

(5

was mi-

a Nissl staining

microinjections

the nuclei,

were

the same conditions

for at least 24 hours.

or when

within

sis was bilateral

blue 8GX)

under

of the tip of the needle

ered a microinfusion third ventricle

Alcian

nuclei

animals

IP) and xylazine

with saline and 9% formaldehyde

in 20% sucrose

ally placed

completed,

(75 mg/kg,

The brain

42 pm were examined

nucleus

were

IP), and a dye (0.05%

croinfused

correct thered

based on previous and noradrenergic

with ketamine

<200

that at

tested with drug pretreatments. Previous experiments indicate that such a colonic response to CRF is indicative of

doses

Brain Histology anesthetized

was

rat) or vehicle

to one pellet.

vehicle

response

tion of CRF (0.6 nmol/rat) time

showed

blockade on CRF microinjected

groups

for their

in a nonstressful

or CRF into the same

two microinjections

into the PVN. Separate

the

time and fecal out-

values of colonic

doses assigned

between

through

rats were re-

experiments

each rat received

was at least 4 days. Effects of autonomic

transit

colon

transit

3 mm

0.2 mL of car-

injections,

of vehicle

reproducible

In this experiment,

ment were tested

saline.

After

deliv-

a 33-gauge

lowered

to their home cages and maintained

Synthetic

by Dr. J. Rivier,

through

Roanoke,

least four microinjections

provided

each

or

restrained

was 100 nL/site

1 minute

Products,

the PVN

lightly

the end of the guide cannula.

put over 24 hours. La Jolla, CA) was dissolved

into

in conscious,

rats. The volume

environment rat CRF (kindly

bilaterally

amygdala

ered successively

turned

awake rats.

in about 40%

Design

was microinfused

the central

exteriorized

and experiments

6-7 days later in nonfasted

717

Effect of CRF microinjected into the PVN and central amygdala. Vehicle (0.1% BSA in saline) or CRF (0.06-0.6 into

used for guide

ROLE OF CRF IN PVN

rats and in the PVN

Experimental

Drugs

0.15

solved

sur-

Products,

from the atlas of Paxinos

steel screws

The Salk Institute, before

(Plastic

coordinates

were individually

were performed

the abdominal

3 mm above the PVN or the

were derived

and stainless

it was se-

STRESS:

80% of implanted

was

and routed

where

cannula

The stereotaxic

placements

suture

AND

of cases.

colon

The catheter

region

Following guide

VA) was implanted amygdala.

cannula ment

skin.

bilateral

1.2 mm; OD,

into the proximal

wall by a purse-string

gery, a 26-gauge Roanoke,

(ID,

junction.

to the interscapular

about

IP, Mobay Corpora-

catheter

TRANSIT

the

our condi-

tions of microinjections. Histological evaluation confirmed a correct bilateral placement in the central amygdala in

Effect of CRF antagonist microinjection into the PVN in rats exposed to water avoidance stress. Separate groups of rats were first tested (0.6 nmol/rat)

for their

microinjected

colonic

into

response

the PVN.

to CRF

A few days

later, the responder as well as 6 nonresponder rats were microinjected bilaterally into the PVN with vehicle (250 nL/site) or the CRF antagonist, a-helical-CRF,,, (13 nmol/rat in 250 nL/site). The volume used for the microinfusion of CRF antagonist into the PVN was the lowest to ensure proper delivery of the peptide in solution. Carmine red (0.2 mL) was injected into the proximal colon 15 minutes after PVN microinjection, and animals were immediately placed in home plastic cage on a plastic cube (dimen-

718

MtiNNlKES

ET AL.

GASTROENTEROLOGY

Vol. 104,

No. 3

sions: height, 6 cm; length, 8 cm; and width, 6 cm) surrounded by water (depth about 3 cm). To avoid contact with the aversive stimulus (water) rats stood on the cube throughout the experiment. Colonic transit time and fecal output were monitored for 24 hours.

Statistical

Analysis

Results are expressed as mean & SEM. The data were analyzed by ANOVA and differences between groups were evaluated by a Mann-Whitney two sample test. P < 0.05 was considered significant. 9 -

Results Effect of CRF Microinjection Into the PVN and Central Amygdala on Colonic Motor Function In PVN,

rats

microinjected

the colonic

transit

and the number hours

of CRF

influence nmol

the PVN

transit transit

in conscious

for the

output

2-hour

did not

doses of 0.2 and 0.6

postmicroinjection

group

1.2 in groups

microinjected

from

showed

stimulation

of fecal output

episodes

following

doses and lasted for 2 hours

ii

0 a+,15

0

were

with CRF into the PVN at respectively

of diarrhea. was observed

(Figure

one third

of

The onset of within

11

0.06

CRF administration

at all

(Table

fecal

1). Thereafter,

was not significantly

be-

groups

(Ta-

ble 1). By contrast, bilateral microinjection of CRF nmol) into the central amygdala did not influence

(0.6 co-

and CRF- (all doses) treated

0.2

0.6

*e

0

0.6

CRF (nmol/rat) Figure 1. Effects of bilateral microinfusion of CRF into the central amygdala on colonic transit time and fecal conscious fed rats. Bars represent means f SEM of indicated at the bottom. *P < 0.001 compared with group.

the PVN or into pellet output in number of rats vehicle-treated

the

different

ionic transit and fecal pellet output (Figure 1). The specificity of CRF action in the PVN to influence coionic transit was further emphasized by the lack of effect of bilateral microinfusions of CRF (0.6 nmol) into sites adjacent to the PVN, namely the zona incerta, the bed nucleus of stria terminalis, the anterior hypothalamus, and the ventromedial hypothalamus (Table 2).

longer

3-

0.4 + 0.3 in the ve-

CRF dose (0.6 nmol),

the animals

tween vehicle-

38. :

of fecal pelperiod

*

Z

to 0.6 * 0.2, 5.7 + 1.3, and 8.3 +

doses of 0.06, 0.2, and 0.6 nmol

output/hour

2

time by 76% and 84% respec-

increased

1). At the highest

the

microin-

at 0.06 nmol

whereas

hicle-treated

pellet

into

for the first

fed rats. The numbers

dose-dependently

first 60 minutes

vehicle

481 + 64 minutes,

was 0.4 + 0.3. Bilateral

into

colonic

decreased

tively, lets

of fecal pellet

post injection

jections

with

time was

with

chlorisondamine

(3 mg’kg,

IP) (Figure

2). Val-

ues of transit time in both vehicle- and CRF-pretreated with chlorisondamine were doubled compared with nonpretreated

group

(Figure

2). Chlorisondamine

treatment also blocked the fecal pellet output hours in both vehicle- and CRF-injected groups; after, there was a trend the CRF-treated

group

a

prefor 6 there-

to higher

fecal output

values in

(Figure

3). Atropine

methyl

nitrate (0.1 mg/kg, IP, 15 minutes before CRF) completely abolished the stimulation of colonic transit and fecal pellet output (Figure 2) induced by CRF microinjetted into the PVN (0.6 nmol). In contrast, a noradrenergic blockade induced by bretylium (15.8 mg/kg,

Effect of Autonomic Blockade on CRF Into the PVN-Induced Stimulation of Colonic Motor Function

IP, 15 minutes before CRF) did not alter the stimulatory effect of CRF on colonic motor function (Figure 2). Neither atropine methyl nitrate nor bretylium pretreatment significantly modified colonic transit time atropine, 448 f 74 min(vehicle, 460 +- 62 minutes;

CRF (0.6 nmol) microinjected stimulated colonic transit

utes; bretylium, 458 + 68 minutes) put (vehicle, 1.4 t 0.5 nb/2 hours;

into the PVN no in rats pretreated

or fecal pellet outatropine, 0.3 + 0.2

March 1993

COLONIC TRANSIT AND STRESS: ROLE OF CRF IN PVN

719

Table 1. Increase in Fecal Pellet Output in Response to Bilateral Microinfusion of CRF Into the PVN in Conscious Fed Rats Pellet output (rib/h))) Dose (nmol/rat)

Treatmenta

no. of rats

Vehicle

CRF CRF CRF

0.06 0.2 0.6

2nd

1st

3rd

13

0.4 f 0.3

0.1 * 0.0

1.5 k 0.6

7

0.6 f 0.2

0.6 k 0.2

2.9 + 1.0

14

2.9 + 0.8’

3.2 k 0.9”

0.8 + 0.2

15

6.0 f

4.0 t 0.8”

2.0 Ik 0.7

1.0”

Yonscious rats chronically implanted with bilateral guide cannulae in the PVN and a catheter in the proximal colon were injected into the PVN with vehicle or various doses of CRF. Pellet output was measured for 3 h post treatment. bMean + SEM. “P < 0.01.

nb/2

hours;

bretylium,

cle-microinjected

1.8 + 1.1 nb/2

rats (Figure

hours)

in vehi-

2).

nificant

decrease

minutes)

(Figure

pellet output

Effect of Psychological Stress and CRF Antagonist in the PVN on Colonic Motor Function In with

nonstressed

vehicle

the number 0.7 + 0.4/2

into

rats

of fecal pellets hours,

microinjected

the PVN,

colonic

treatment

transit

4). Stress-induced

was also completely

with

a-helical-CRF,,,

Six rats, in which did not induce

increase

CRF

time

increase inhibited (Figure

microinjection

colonic

(346

transit,

+ 27

in fecal

by the pre4). (0.6 nmol)

were microin-

bilaterally time

and

were 470 Ifr 72 minutes

and

respectively.

in colonic

transit

The CRF antagonist

(13 nmol) microinjected bilaterally into the PVN did not alter significantly the colonic transit time (407 + 88 minutes) and fecal pellet output in rats maintained in a nonstressful ure 4). Exposure the colonic and increased

transit

to water

avoidance

hours) (Fig-

stress decreased

time by 75% in vehicle-treated

fecal pellet

output

first 2 hours of stress exposure croinjected with a-helical-CRF,,, nmol),

(2.4 k 0.5/2 environment

water avoidance

by 7-fold

rats

during

the

(Figure 4). In rats miinto the PVN (13

stress no longer

induced

a sig-

Table 2. Influence of CRF Microinjected

Outside of the PVN on Colonic Transit Time and Fecal Pellet in Conscious Rats

Treatmenta

no. of rats

Vehicle outside PVN CRF Zona incerta CRF BNST

17 3 2

CRF AHN

6

CRF VMH CRF Others

2 3

Transit time (mi# 443 * 75 400 f 127 371 f 101 398 rf: 171 411 + 142 396 + 206

Fecal output (W2 1.8 2.3 2.0 2.8 2.1 2.7

+ f * + + +

h) 1.5 0.9 1.0 1.5 2.0 1.8

Yonscious rats chronically implanted with bilateral cannulae and a catheter in the proximal colon were injected with CRF (0.6 nmol/rat) in various hypothalamic sites (BNST, Bed Nucleus Stria Terminalis; AHN, Anterior Hypothalamus Nucleus; VMH, Ventromedial Hypothalamus. Others: medial preoptic area, nucleus reunien, anterior commissural nucleus, 1 each) and carmine red in the proximal colon. Colonic transit time and fecal pellet output were monitored post injection. bMean -e SEM.

(w/kg

I.p.1

(3)

(0.10)

(15.8)

Figure 2. Effects of chlorisondamine, atropine, or bretylium on CRF microinjected into the PVN-induced stimulation of colonic motor function in conscious fed rats. Pretreatments were given 15 minutes before microinjection of CRF or vehicle into the PVN. Bars represent means + SEM of the number of rats indicated at the bottom. *P < 0.0 1 compared with respective vehicle into the PVN treated groups. ‘P < 0.01 compared with vehicle-vehicle treated group. 0. Vehicle into PVN; ?? , CRF. 0.06 nmol into PVN.

MijNNlKES

ET AL.

GASTROENTEROLOGY

Vol. 104,

No. 3

18 15

3. Kinetic of cumulated fecal pellet output in response to CRF into the PVN in chlorisondamine-treated rats. Data are means 2 SEM (n = 5 in each group). *P < 0.05 compared with the vehicles-treated group. A, Vehicle, IP and CRF, 0.6 nmol into PVN; A, vehicle, IP and vehicle into PVN; ?? , chlorisondamine, 0.33 mg/kg IP and CRF, 0.6 nmol into PVN; 0, chlorisondamine, 0.33 mg/ kg IP and vehicle into PVN. Figure

12 T

T

T

9 6 3 0 0

2

4

6

8

jetted with the CRF antagonist conditions,

10

12

(13 nmol).

14

16

Under

these

there was a slight nonsignificant

in the decrease

in colonic

transit

reduction

time induced

by pas-

sive avoidance stress (vehicle plus stress: 116 + 21 minutes, n = 14; CRF antagonist outside of the PVN plus stress: 229 * 52 minutes, the stimulation

of fecal pellet

sive avoidance

stress (vehicle

hours;

CRF antagonist

4.1 f

0.9/2

croinjection

n = 6) and no changes

hours).

output

by pas-

plus stress: 4.9 + 0.6/2

outside

of the PVN plus stress:

Histological

sites after

induced

in

examination

the experiments

of mi-

showed

that

18

20

22

episodes

24

of diarrhea

in one third

expulsion

of the liquid

content

ionic

transit

time appears

crease in fecal pellet output 2 hours. inducing

maximal

ent observation)

(0.6 nmol)

the lateral

ventricle

zona incerta and medial

(n = 1), anterior preoptic

nucleus

stria terminalis

(n = l),

hypothalamus

(n = 2),

(n = 1).

Our results

extend

of colonic

previously

transit

ways in the PVN as mediators to psychological

brain

stimulation

published

obser-

CRF in stress-related

by identifying of the colonic

CRF pathresponse

stress.

CRF injected into the lateral ventricle or the PVN was previously reported to stimulate colonic transit measured within 60 minutes following peptide injection by the geometric center method in conscious fasted rats.3,6,8,13 In the present study, microinjection of CRF into the PVN stimulated colonic transit and fecal pellet output as measured by the time interval (80-480 minutes) between dye injection into the proximal colon and discharge of colored pellet in conscious fed rats. Microinjection of CRF into the PVN at 0.6 nmol reduced the duration of colonic transit time by 85%, increased fecal pellet output by 20-fold and induced

(1-2

the dose of CRF

of colonic

transit

is

into the PVN13 (pres-

than

of colonic

the in-

for at least

when

delivered

into

nmo1).3*6 transit

and fecal pellet

output induced by CRF microinjected into the PVN may be related to propulsive changes in colonic motilPVN at 0.6 nmol

vation&8*13 for the role of central stimulation

The

ity, In a preliminary

Discussion

studies,

stimulation

had been microinjected

(n = 1), bed nucleus

colon

because

was maintained

lower when CRF is microinjected

namely

reuniens

of the proximal

long lasting

Based on previous

CRF and the CRF antagonist

into the nucleus

The

and/or alterations of secretory and absorptive processes in the intestine.” CRF stimulatory effect on co-

into sites outside

of the PVN

of the animals.

appearance of diarrhealike feces after microinjection of CRF at the highest dose into the PVN may reflect

and the amplitude mal colon

study, CRF microinjected increased of phasic

in fasted

rats.”

tonic intraluminal contractions In addition,

into the pressure

in the proxiCRF

injected

into the lateral ventricle has been reported to increase spike-burst frequency in the cecum and proximal colon in fasted

rats.5*21

CRF-induced stimulation of colonic motor function appears specific to the PVN because microinjection of the peptide into other hypothalamic sites outside of the PVN (zona incerta, bed nucleus stria terminalis, anterior hypothalamus, and ventromedial hypothalamus) and into the central amygdala did not alter coionic transit. These data, along with our previous report,13 provide the first evidence that the PVN may play an important role in the regulation of colonic motility in rats. Previous studies indicate that electrical stimulation of the lateral, ventromedial, or anterior hypothalamus can influence cecal, colonic, or rectal

March

COLONIC

1993

r-5

TRANSIT

AND

In a previous

vagotomy prevented, by only 36%, of colonic transit induced by CRF mi-

into

these

the PVN

data

CRF in the PVN

volves

peripheral

duced

hexamethonium excitatory

tor activity atropine The

I

A 2 F

o-

r-l

4

5

14

CONTROL

11

Figure 4. Effect of CRF antagonist microinjection into the PVN on passive-avoidance stress (PA)-induced stimulation of colonic motor function in conscious fed rats. ‘P < 0.001 compared with a-helicalCRF,_,, PA group. *P < 0.01 compared with control-vehicle group. The data are means k SEM of the number of rats indicated at the bottom of each bar. 0, Vehicle; W a = helical CRF,_,, , 13 nmol.

in anesthetized

cholecystokinin medial burst

(CCK)

rats or cats22,23 and that

microinjected

hypothalamus

increases

into the ventrocecocolonic

spike

in fasted rats.24

The complete fect on colonic

nicotinic blockade that peptide action

of the CRF stimulatory

and fecal output

ef-

by ganglionic

using chlorisondamine may be mediated through

indicates the auto-

relevance

and

transit,

showing

the cerebrospinal

into

or frequency

fasted

rats.5,6*8 Stress activity

room

temperature

that CRF in the PVN does not

is known

spike

fecal

the autonomic

pellet

output.

Restraint

activation spike

stimulates

of

the

is mediated

the frequency fasted

transit

of the colonic

of

or fed stress of

space to avoid an aversive colonic

at

tran-

pituitary-adrenal

in conscious

in fed rats as previously

a1.15 The magnitude

in rats, colonic

effect

in

colonic

study, the psychological

on a confined

colonic

system and is indepen-

fear increased

bursts

rats.5*21 In the present

This

nervous

activ-

bursts

to stimulate

or in cold, stimulated

sit and

cecocolonic

motor

of a-helical-CRF,,,

in humans.29,30

the

Under

did not modify

of cecocolonic

through of

a-he-

the PVN.

fluid did not influence

transit motor

in the

using the CRF antagonist,

injection

stim-

and fecal pellet

reported motor

by Enck response

et was

similar to that observed with CRF microinjected into the PVN at the 0.2 nmol dose. In addition, bilateral

axis to these effects is unlikely.

induced

peripheral cholinergic muscarinic pathways is supported by the inhibition of CRF action by atropine methyl nitrate, whereas noradrenergic blockade with bretylium had no effect on CRF action. These findings are consistent with a previous report showing that intracerebroventricular CRF-induced stimulation of large bowel transit in fasted rats is abolished by the ganglionic blocker chlorisondamine and is not modified by hypophysectomy, adrenalectomy, bretylium,

of a mo-

action

ity in fed rats. Similarly,

pletely

through

of CRF

action on colonic

output

in fed

on colonic

exert a tonic stimulatory

nomic nervous system. These data also suggest that a contribution of the hypothalamic-pituitary-adrenal A mediation

influence

the CRF antagonist

ulus (water)

inhibition transit

observed

blocker

to the removal

basal conditions,

standing

in-

in fasted and fed monkeys.26

axis.6,8,25,31Conditioned contractility

the The

output

was previously

microinjected

dent

This

through

and pellet

lical-CRF,_4,27,28

into

PA-STRESS

in-

using vagal cryointerruption

physiological

colonic

3-

transit

cholinergic

as shown

by

transit

pathways.

may be related

PVN was investigated

I

mediated

as a ganglionic

rats.25 The decrease tonic

colonic

neurotransmission.

by chlorisondamine

using

to-

mechanism

parasympathetic

in basal colonic

that

rats.13 Taken

the

influences

is probably

of sacral

decreases

that

cholinergic

neurotransmission

we showed

in fasted

suggest

which

activation

study,

721

and naloxone.3

croinjected

i#

ROLE OF CRF IN PVN

subdiaphragmatic the stimulation gether,

N. S.

STRESS:

microinjection

of CRF antagonist

prevented by water

the

increase

avoidance

into the PVN comin fecal-pellet

output

stress and normalized

the

values of transit time. The specificity of CRF antagonist action in the PVN was shown by the lack of a similar inhibitory effect of the antagonist injected in sites adjacent to the PVN. These neuropharmacological observations indicate that endogenous CRF in the PVN may be directly involved in the central nervous system mechanisms through which various stressors increase colonic motor activity. A physiological role of CRF in the PVN to

722

MiiNNlKES ET AL.

mediate

colonic

GASTROENTEROLOGY Vol. 104, No. 3

response

by the demonstration CRF-containing increase

that

neurons

various

In summary, propulsive

colonic

rats. In addition,

function

the physiological

microinjected

avoidance

into

Taken

function

stress.

during

of fed

of CRF in

to prevent

passive

of colonic

mo-

these data indicate

that

may be an important in mediating

is a

of the CRF antago-

stimulation

for CRF action

the PVN stimulation

relevance

the PVN

together,

in an

in conscious

by the ability

stress-induced

the PVN

that

neuroanatomic alterations

paraventricular nucleus mediates gastric and colonic motor response to restraint stress. Am J Physiol 1992;262:G 137-G 143.

activate

in this nucleus.32-34

show

motor

supported

and result

for CRF-induced

the PVN is indicated

tor function.

RNA

the results

site of action

stressors

in the PVN

of CRF messenger

specific

nist

to stress is further

locus

of gut motor

14. Tache Y, Goto Y, Gunion MW, Vale W, Rivier J, Brown M. Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor. Science 1983;222:935-937. 15. Enck P, Merlin V, Erckenbrecht JF, Wienbeck M. Stress effects on gastrointestinal transit in the rat. Gut 1989;30:455-459. 16. Gray TS, Magnuson DJ. Neuropeptide neuronal efferents from the bed nucleus of the stria terminalis and central amygdaloid nucleus to the dorsal vagal complex in the rat. J Comp Neurol 1987;262:365-374. 17. Paxinos G, Watson C. The rat brain in stereotaxic Orlando: Academic, 1986. 18. Yanagisawa K, Tache Y. lntracisternal stimulates gastric histamine release 1990;259:G599-G604.

,

1. Lenz HJ. Stress-induced

alteration of gastrointestinal function: role of corticotropin-releasing factor. In: Tache Y, Wingate D, eds. Boca Raton: CRC, 199 1:285-295.

2. B&no L. Role of corticotropin-releasing factor in the genesis of gastrointestinal motor disturbances induced by stress: an overview. In: Buena L, Collins S, Junien JL, eds. Stress and Digestive Motility. Montrouge: John Libbey Eurotext, 1989: 14 1- 149.

TRH analog RX 77368 in rats. Am J Physiol

19. Lenz HJ. Regulation of duodenal bicarbonate secretion during StreSS by corticotropin-releasing factor and j3-endorphin. Proc Natl Acad Sci USA 1989;86: 14 17- 1420. 20.

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Received July 16, 1991. Accepted October 6, 1992. Address requests for reprints to: Yvette Tache, Ph.D., Center for Ulcer Research and Education, Veteran’s Affalrs Wadsworth Medical Center, Building 115, Room 203, Wilshire and Sawtelle Boulevard, Los Angeles, California 90073. Supported by the National Institute of Arthritis, Metabolism and

COLONIC TRANSIT AND STRESS: ROLE OF CRF IN PVN

Digestive

Disease, grant DK-33061,

and the National Institute

723

of

Mental Health, grant MH-00663. Hubert Monnikes received grants from the Deutsche Forschungsgemelnschaft, and Kempkes-Stlftung, (Marburg) Germany. The authors thank Dr. Jean Rivier of The Salk Institute, La Jolla, California, for his generous donation of rat corticotropin releasing factor. A preliminary report of this study was presented at the annual meeting of the American Gastroenterological Association and was published in abstract form (Gastroenterology 1991;100:A656). The authors thank Paul Kirshbaum for editing the manuscript.